More specifically, this invention relates to the biosensors that are used to measure the amount of analytes in bodily fluids, particularly measurements of glucose in samples of whole blood. Optical methods are often used for making such measurements, but the present invention relates to improvements in electrochemical biosensors.
While the method of the invention to be described herein can be applied to measurement of other analytes, including cholesterol, urea, creatinine, and creatine, measuring glucose in whole blood is of particular interest. The invention relates to an electrochemical instrument in which a constant or varying potential is applied to electrodes in contact with a blood sample and the resulting current is measured over a short period of time and then correlated with the amount of an analyte in the sample. Such instruments are referred to as amperometric, in contrast with instruments that measure the total current produced from reaction of the sample and are referred to as coulometric. The amperometric instruments have an advantage in that they carry out their test measurement within a short time compared to those in which the total current produced in oxidizing a sample is measured.
Glucose biosensors of the amperometric type measure the current produced when a fixed potential is applied across a pair of electrodes in contact with a sample of blood. The measured current begins at a high value and then declines and approaches a constant value related to the diffusion of a reduced mediator compound to one of the electrodes for re-oxidation. At a predetermined time, the measured current is used to determine the glucose content of the sample.
The electrodes are generally described as the working electrode (i.e., the electrode at which the mediator is oxidized) and as the counter electrode. Many designs for such biosensors have been described in the art, for example, published U.S. Pat. No. 6,531,040. The electrodes are in contact with a solid layer containing reagents that oxidize the glucose in the sample, such as glucose oxidase and mediators that reoxidize the reduced enzyme. The reduced mediator itself is reoxidized at the working electrode as described above, thereby producing a measurable current, which had been previously correlated with the amount of glucose in the sample being tested. The reactions may be described by the following steps:Glucose+Eoxid→Ered+Oxidized Glucose (Gluconolactone)Ered+n Medoxid→n Medred+Eoxid n Medred→Medoxid+n e−
Where Eoxid and Ered are oxidized and reduced forms of the redox center of the enzyme and Medoxid and Medred are the oxidized and reduced forms of the mediator.
For measuring glucose, the enzyme may be glucose oxidase and the mediator ferricyanide. Measuring other analytes will employ suitable enzymes and mediators. For example, cholesterol may be measured using cholesterol esterase and ferricyanide, while alcohol may be measured using alcohol oxidase and phenylenediamine. Typical combinations of enzyme, mediator and analyte are listed in Table 1.
TABLE 1AnalyteEnzymeMediatorGlucoseGlucose OxidaseFerricyanideGlucoseGlucose DehydrogenaseFerricyanideCholesterolCholesterol OxidaseFerricyanideLactateLactate OxidaseFerricyanideUric AcidUricaseFerricyanideAlcoholAlcohol OxidasePhenylenediamine
The reagents are supplied in larger amounts than are required in order to make glucose in the blood sample the limiting reaction constituent. It is important that the amount of blood in the sensor be substantially the same from one sensor to another and that each sensor be uniformly filled. But, it has been found that underfilling of biosensors is frequent enough to present a significant problem in assuring consistent and accurate measurements of blood glucose. Clearly, if a person with a diabetic condition must make frequent measurements of his or her blood glucose, it is vital that those measurements be accurate and reliable. Therefore, it is desirable that an amperometric instrument be capable of detecting when a biosensor has been underfilled and is providing an incorrect result, so that the result can be discarded and the test repeated, or the measured current can be adjusted by another algorithm.
Another problem, which was addressed in U.S. Pat. Nos. 5,620,579 and 5,653,863, relates to the premature reducing of the mediator during the period of shelf life before the biosensor is used. If a sample is applied to such a sensor, the reduced mediator will be reoxidized at the working electrode, making it appear that additional glucose was present in the sample, thus giving an incorrect high value. The patentees proposed to begin the test of a sample by providing an initial positive potential pulse for a short period in order to reoxidize any prematurely reduced mediator. Such an initial pulse was referred to as a “burnoff period”. The patentees further proposed a method of correcting for the bias introduced by reduced mediator in the sensor. The present invention addresses the data obtained in the burn period as they relate to the filling of biosensors.
In U.S. Pat. No. 6,531,040, an improved amperometric biosensor was described. In one aspect, the biosensor was intended to provide a signal indicating that incomplete filling by the sample had occurred. This was to be accomplished by providing a sub-element of the counter electrode upstream of the working electrode, that is, the sample as it flowed into the sensor by capillary action first reached the working electrode and then the counter electrode. When an underfill condition occurred, the current would be much weaker than would normally be expected and would be recognized as indicating that an underfill had occurred. Another method was described that used measurements of the current during the so-called “read” and “burn” periods to predict that an underfill had occurred. Such methods were also described in U.S. Patent Application Publication No. 2004/0154932.
A general description of the plots of current versus time generated when a constant potential is applied across working and counter electrodes in an amperometric sensor may be helpful to the reader.
In general, when a potential is applied across the working and counter electrodes and a liquid sample of blood or control solution is introduced to the sensor, the dry reagents are rehydrated by the liquid sample and current begins to flow, typically increasing to a peak and then declining over the “burn period,” usually about ten seconds in length. During this period the previously reduced mediator is reoxidized, as discussed above, to reduce the bias towards falsely elevated values of glucose content. If a full amount of sample is not present, additional error may be introduced since all of the reagents may not become available for reaction or the working and counter electrodes might not be in complete contact with sample, thus reducing the current during the “burn” period.
After the burn period has been completed, a rest period is provided at a lower potential or at no potential during which the glucose oxidation reactions take place and the mediator is reduced. Then, a constant potential is applied again between the working and counter electrodes and the current is measured for a short period, typically about ten seconds. The current is initially high, but it declines and approaches a constant value that is used to determine the glucose content of the sample. In the methods of the published application and the issued patent referred to above values of the current are taken at certain times in the burn and read periods and used to predict underfilling of the sensor. However, improved methods of predicting underfilling have been sought.
The present inventors have found an improved method of determining underfilling of sensors which will be described below.